The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway¹

Abstract

MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined. The three-dimensional structure of the Escherichia coli protein was solved at 2.2 Å resolution. The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site. Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog. Domain 3, in contrast, is structurally similar to many other proteins. The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis. The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions.

Introduction

Molybdenum is an essential trace metal in most living organisms. With the exception of nitrogenase, all of the known Mo-dependent enzymes in bacteria, archaea and eukaryotes use a molybdenum cofactor (Moco) consisting of a molybdopterin (MPT) compound that coordinates a molybdenum atom via a dithiolene moiety.1, 2 The Moco is synthesized in a multi-step synthetic pathway that is conserved among eubacteria, archaea and eukaryotes.1, 3, 4 The genes involved in Moco synthesis were identified by mutations that resulted in chlorate resistance and the genetic loci were first known as chl loci.1 As their role in molybdopterin synthesis became clear, the nomenclature was changed to the mo loci.5 Mutations in the mo loci cause pleiotropic losses of the activities of all Mo-dependent enzymes in the organism. The cofactor is extremely labile when not bound by protein and has not been isolated in a pure form.1, 6, 7

The biosynthesis of molybdopterin can be divided into three stages; synthesis of precursor Z (probably from GTP), formation of the dithiolene group, and insertion of the molybdenum atom (Figure 1). 2, 6, 7 For some prokaryotes, an additional step involving fusion of a dinucleotide to the cofactor side-chain is also required. Guanine, cytosine, adenine and hypoxanthine dinucleotide forms of the Moco have been identified.1 Molybdo-enzymes in Escherichia coli require the guanine dinucleotide form (MGD).

Complex mechanisms have evolved to transport, synthesize, regulate and stabilize the molybdopterin cofactor. There are five operons in E. coli to accomplish those tasks: mod, moa, mob, mog and moe.2 The functions of some of these operons are well understood and others are being actively investigated. The mod operon constitutes a molybdate transport system related to the ATP-binding cassette (ABC) transporter superfamily and regulates the expression of the mod genes in a Mo-dependent fashion.9, 10, 11, 12 The MoaA and C proteins synthesize the molybdopterin precursor8 and MoaD and E, also known as molybdopterin synthase, incorporate the sulfur atoms into the dithiolene to form MPT.13, 14, 15, 16 MoeB, also known as MPT synthase sulfurylase, provides the sulfur atoms that are transferred by MPT synthase to the dithiolene group of MPT.14, 15 E. coli Mo-dependent enzymes use molybdopterin guanine dinucleotide (MGD), a derivative of MPT with a GMP molecule fused to MPT. MobA catalyzes the reaction between GTP and MPT to form MGD.17, 18, 19 The functions of the mog and moe operons are, on the other hand, less well understood. MogA is reportedly a molybdochelatase responsible for insertion of the Mo atom into molybdopterin to complete Moco synthesis,20 but the mechanism of this transfer is not known. The function of MoeA is the least certain, but several possibilities have been suggested. MoeA has been suggested to be necessary for Mo insertion into MPT,21 for formation of activated thiomolybdate compounds, which are then used by MogA to transfer the Mo atom to MPT,22 and for formation of a scaffold on which Moco synthesis occurs.23

MoeA and MogA homologs are fused in mammalian gephyrins,24, 25 Drosophila cinnamon,26 and Cnx1 from Arabidopsis.4 Fusions in higher organisms of genes expressed separately in bacteria and archaea may indicate that the two proteins of the fusion interact to accomplish their biological function.27 Although the interaction between MogA and MoeA has been postulated by several investigators,4, 24, 28 there is no substantial evidence to indicate that it occurs in bacteria. The three-dimensional structure of MoeA presented here reveals structural similarities between MoeA and MogA. Although no definite biochemical role for MoeA derives directly from the structure, the location of its active site can be identified from the location of a cluster of conserved amino acid residues within a deep indentation in the enzyme surface. Structural similarities to MogA suggest the possibility that the two proteins bind similar ligands, consistent with the suggestion that they interact and work together in the final stage of Moco synthesis.